title: Modification of tyrosine residues in dihydrofolate reductase from Lactobacillus casei.
abstract: Dihydrofolate reductase from Lactobacillus casei was inactivated by reaction with tetranitromethane and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Loss of activity occurred with modification of four of the five tyrosine residues present in the enzyme. The presence of either substrate, NADPH or 7,8-dihydrofolate, as well as NADP and folate, provided extensive protection against inactivation, while NADH and tetrahydrofolate exhibited none. This protection from inactivation occurred on protection of two of the four susceptible tyrosines from modification. Nitration of the enzyme adversely affected its ability to bind substrates. Restoration of the pKa of the nitrated tyrosines by reduction of the nitro group to an amino group did not result in a regeneration of enzymatic activity. However, fluorotyrosine-containing enzyme, prepared by growing the bacterium in the presence of fluorotyrosine, exhibited specific activity identical to that of native enzyme over the pH range of 4.5-8. These results suggest that inactivation of dihydrofolate reductase by tyrosine modification occurs primarily due to a steric effect and that the active site tyrosines may participate in substrate binding.
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005492:Folic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D009249:NADP / Q000494:pharmacology; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives
chemical_list: C001245:3-fluorotyrosine; C010920:dihydrofolate; D014443:Tyrosine; D009249:NADP; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D009327:4-Chloro-7-nitrobenzofurazan; D013774:Tetranitromethane